Machine learning-driven exosome-mimetic lipid nanoparticles for tumor-specific targeting.

The study aims to investigate the ability of lyophilized nasal inserts of nanosized atomoxetine HCl solid lipid nanoparticles (ATM-SLNs) to transport atomoxetine (ATM) directly to the brain and overcome the first-pass metabolism. In this case, 16 formulae of (ATM-SLNs) were prepared using hot melt emulsification, stirring and ultrasonication method technique. A full factorial design was established with 24 trials by optimization of four variables; lipid type (Compritol 888 ATO or stearic acid) (X1), lipid to drug ratio [(1:2) or (2:1)] (X2), span 60: Pluronic f127 ratio [(1:3) or (3:1)] (X3) and probe sonication time (five or ten minutes) (X4). The prepared SLNs were characterized for entrapment efficiency (EE%), in-vitro drug release after 30 min (Q30min), particle size (PS), zeta potential (ZP) and polydispersity index (PDI). Design Expert® software was used to select the optimum two formulae. The morphological examination for the optimum two formulae was carried out using a transmission electron microscope (TEM). Furthermore, eight lyophilized nasal inserts were prepared by using a 23 full factorial design by optimization of three variables: type of (ATM-SLNs) formula (X1), type of polymer (NOVEON AA1 or HPMC K100m) (X2) and concentration of polymer (X3). They were evaluated for nasal inserts' physicochemical properties. The two optimum inserts were selected by Design Expert® software. The two optimum insets with the highest desirability values were (S4 and S8). They were subjected to DSC thermal stability study and in-vivo study on rats. They were compared with atomoxetine oral solution, atomoxetine (3 mg/kg, intraperitoneal injection) and the pure atomoxetine solution loaded in lyophilized insert. (ATM-SLNs) showed EE% range of (41.14 mg ± 1.8% to 90.6 mg ± 2.8%), (Q30min%) of (27.11 ± 5.9% to 91.08 ± 0.15%), ZP of (-8.52 ± 0.75 to -28.4 ± 0.212% mV), PS of (320.9 ± 110.81% nm to 936.7 ± 229.6% nm) and PDI of (0.222 ± 0.132% to 0.658 ± 0.03%). Additionally, the two optimum (ATM-SLNs) formulae chosen, i.e., F7 and F9 showed spherical morphology. Nasal inserts had assay of drug content of (82.5 ± 2.5% to 103.94 ± 3.94%), Q15min% of (89.9 ± 6.4% to 100%) and Muco-adhesion strength of (3510.5 ± 140.21 to 9319.5 ± 39.425). DSC results of S4 and S8 showed compatibility of (ATM) with the other excipients. S8 and S4 also showed higher trans-nasal permeation to the brain with brain targeting efficiency of (211.3% and 177.42%, respectively) and drug transport percentages of (52.7% and 43.64%, respectively). To conclude, lyophilized nasal inserts of (ATM-SLNs) enhanced (ATM) trans-nasal drug targeting permeation and brain targeting efficiency.

Background/Objectives: Drug repurposing explores new applications for approved medications, such as simvastatin (SV), a lipid-lowering drug that has shown anticancer potential but is limited by solubility and side effects. This study aims to enhance SV delivery and efficacy against lung cancer cells using bioactive lipid nanoparticles formulated with plant-derived monoterpenes as both nanostructuring agents and anticancer molecules. Methods: Lipid nanoparticles were produced by ultrasonication and characterized for morphology, size, zeta potential, and polydispersity index (PDI). Monoterpenes (linalool-LN-, limonene, 1,8-cineole) or Crodamol® were used as liquid lipids. Encapsulation efficiency (EE), release profiles, stability, biocompatibility, protein adsorption, cytotoxicity, and anticancer effects were evaluated. Results: The nanoparticles exhibited high stability, size: 94.2 ± 0.9–144.0 ± 2.6 nm, PDI < 0.3, and zeta potential: −4.5 ± 0.7 to −16.3 ± 0.8 mV. Encapsulation of SV in all formulations enhanced cytotoxicity against A549 lung cancer cells, with NLC/LN/SV showing the highest activity and being chosen for further investigation. Sustained SV release over 72 h and EE > 95% was observed for NLC/LN/SV. SAXS/WAXS analysis revealed that LN altered the crystallographic structure of nanoparticles. NLC/LN/SV demonstrated excellent biocompatibility and developed a thin serum protein corona in vitro. Cellular studies showed efficient uptake by A549 cells, G0/G1 arrest, mitochondrial hyperpolarization, reactive oxygen species production, and enhanced cell death compared to free SV. NLC/LN/SV more effectively inhibited cancer cell migration than free SV. Conclusions: NLC/LN/SV represents a promising nanocarrier for SV repurposing, combining enhanced anticancer activity, biocompatibility, and sustained stability for potential lung cancer therapy.

We report the formulation of aminocellulose-grafted polymeric nanoparticles containing LCS-1 for synthetic lethal targeting of checkpoint kinase 2 (CHEK2)-deficient HCT116 colon cancer (CRC) cells to surpass the limitations associated with the solubility of LCS-1 (a superoxide dismutase inhibitor). Aminocellulose (AC), a highly biocompatible and biodegradable hydrophilic polymer, was grafted over polycaprolactone (PCL), and a nanoprecipitation method was employed for formulating nanoparticles containing LCS-1. In this study, we exploited the synthetic lethal interaction between SOD1 and CHEK2 for the specific inhibition of CHEK2-deficient HCT116 CRC cells using LCS-1-loaded PCL-AC NPs. Furthermore, the effects of formation of protein corona on PCL-AC nanoparticles were also assessed in terms of size, cellular uptake, and cell viability. LCS-1-loaded NPs were evaluated for their size, zeta potential, and polydispersity index using a zetasizer, and their morphological characteristics were assessed by transmission electron microscopy, scanning electron microscopy, and atomic force microscopy analyses. Cellular internalization using confocal microscopy exhibited that nanoparticles were uptaken by HCT116 cells. Also, nanoparticles were cytocompatible as they did not induce cytotoxicity in hTERT and HEK-293 cells. The LCS-1-loaded PCL-AC NPs were quite hemocompatible and were 240 times more selective in killing CHEK2-deficient cells as compared to CHEK2-proficient CRC cells. Moreover, PCL-AC NPs exhibited that the protein corona-coated nanoparticles were incubated in the human and fetal bovine sera as visualized by SDS-PAGE. A slight increment in hydrodynamic diameter was observed for corona-coated PCL-AC nanoparticles, and size increment was further confirmed by TEM. Corona-coated PCL-AC NPs also exhibited cellular uptake as demonstrated by flow cytometric analysis and did not cause cytotoxic effects on hTERT cells. The nanoformulation was developed to enhance therapeutic potential of the drug LCS-1 for enhanced lethality of colorectal cancer cells with CHEK2 deficiency.

Influence of ionisable lipid and sterol variations on lipid nanoparticle properties and performance.

Multivariate design for the evaluation of lipid and surfactant composition effect for optimisation of lipid nanoparticles

Enhanced tyrosine kinase inhibitor response on chronic myeloid leukemia cells upon MS4A3 mRNA delivery

Understanding the effect of lipid and surfactant composition on particle size and colloidal stability plays a pivotal role in designing lipid nanoparticles (LN) for drug delivery. With respect to our long-term goal, LN for brain delivery, formulations containing lipids and surfactants suitable for intravenous (i.v.) application were selected for the current formulation screening study. LN were prepared by hot high pressure homogenization (HPH) and were characterized during 1 year in terms of macroscopic appearance, particle size by photon correlation spectroscopy (PCS) and optical single particle sizing (OSPS), zeta potential (ZP), as well as physical state and polymorphism by differential scanning calorimetry (DSC). The LN dispersions showed a wide variability in macroscopic appearance, mean size and colloidal stability. Influence factors were the type and concentration of both, the lipid and surfactant component used. The most promising LN showed a small mean size (< 200 nm), a low polydispersity index (PI), (< 0.25) absence of particles in the several-micron range, and a slightly negative ZP (> −12 mV); DSC revealed that some represented supercooled liquids; such LN may be stable at room temperature for at least 1 year. The obtained results are regarded helpful for defining the design space for LN delivery systems, i.e., identifying possible designs and design parameters within the given HPH technology to be applied during future formulation development studies.

RNA interference is a powerful research tool for studying gene function in cancer cells. However, functional genomic studies in Acute Myeloid Leukemia (AML) remain challenging. Classic lipid-based transfection reagents are ineffective in delivering siRNA to AML cells and lentiviral delivery of shRNA is complex and time-consuming. Lipid Nanoparticles (LNPs) are a novel approach to deliver genetic material to cells that are difficult to transfect. However, a formulation of LNP capable of transfecting AML cells and primary AML samples with high efficiency and low toxicity is lacking. Here, we developed an LNP formulation that delivers siRNA to AML cell lines and primary samples without impacting their growth and viability. To identify LNPs able to transfect AML cell lines, we generated a library of LNPs that differed in lipid compositions, aqueous phase pH, and RNA-to-ionizable lipid weight ratio through the Design of Experiment (DOE). We synthesized 54 unique LNPs formulations encapsulating siRNA targeting luciferase. We then screened this library of LNPs against OCI-AML2 leukemia cells over-expressing luciferase. From this screen and subsequent validation studies, we identified a novel formulation, LNP 1-28, as the top hit based on transfection efficiency and safety. LNP1-28 also suppressed luciferase expression in 3 AML cell lines (OCI-AML2, OCI-AML3, MOLM13) with &amp;gt; 80% target knockdown with 100nM of siRNA 3 days post-transfection. Next, we asked if LNP 1-28 could deliver siRNA to knockdown an endogenous protein, CD45. AML cell lines (OCI-AML2, OCI-AML3, MOLM13) were treated with LNP 1-28 carrying siRNA targeting CD45 or control sequences. Knockdown of CD45 protein was observed by flow cytometry in all tested AML cells with knockdown being time and concentration dependent. CD45 knockdown was observed as early as 1-day post-transfection and persisted for 7 days before gradually returning towards baseline. Greater than 82.5% knockdown was observed 4 days after 100nM siRNA-LNP treatment in all tested cell lines. LNP 1-28 had no effect on cell growth and viability. Using the low passage primary AML culture model 8227 cells, we demonstrated that LNP 1-28 could deliver siRNA to both bulk and CD34+/CD38- stem populations of AML. Finally, LNP 1-28 efficiently transfected primary AML patient samples and delivered siRNA to knock down CD45. In summary, we developed a novel LNP formulation capable of transfecting AML cells and primary AML samples with siRNA at high efficiency and without toxicity. Thus, we have developed a nanocarrier system for in vitro transfection that will aid in functional genomics studies of AML. Citation Format: Lan Xin Zhang, Yongran Yan, Jingan Chen, Rose Hurren, Jiachuan Bu, Juan Chen, Dakai Ling, Nathan Duong, Andrea Arruda, Hansen He, Mark D. Minden, Rod Bremner, Gang Zheng, Bowen Li, Aaron D. Schimmer. A novel lipid nanoparticle formulation transfects acute myeloid leukemia cells and primary AML samples with high efficiency and low toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1792.

Ionizable lipid nanoparticles with functionalized PEG-lipids increase retention in the tumor microenvironment.

Development of a binary lipid nanoparticles formulation of itraconazole for parenteral administration and controlled release

Lornoxicam, is a NSAID of the oxicam class. Its short duration of action owing to rapid elimination and gastrointestinal side effects limits its usefulness when administered orally. The primary objective of the proposed work is to develop suitable lipid nanocarriers for transdermal delivery of Lornoxicam with increased drug residence time at local site of inflamation and in systemic circulation, overcoming undesired gastrointestinal side effects. Lornoxicam loaded lipid nanocarriers like solid lipid nanocarriers (SLN), nano-structured lipid carriers (NLC) & nanoemulsions (NE) were prepared by high-speed homogenization technique. The particle size, zeta potential, and polydispersity index as obtained, were in the range of 140- 193 nm, -22 to -32 mV, and 0.354-0.301 for SLN formulations and 146-201 nm, -23 to -30 mV, and 0.355-0.354 for NLC formulations respectively. Characterization of stable NE revealed that globule size, zeta potential and polydispersity index were within the range of 138 to 195 nm, -26.1±0.123 mV and 0.195 ± 1.231 respectively. It was also observed that entrapment efficacy and drug loading improved as the lipid concentration was increased. The results obtained from the in vitro permeation study and in vivo anti-inflammatory study showed controlled drug permeation, increased bioavailability, longer retention and better therapeutic potential of Lornoxicam after transdermal application of lipid nanoparticles as compared to conventional gel. It can be concluded that the developed lipid nanoparticle loaded gel was found to be a suitable drug delivery carrier for transdermal delivery of Lornoxicam to increase the residence time of drug in systemic circulation and to combat the gastrointestinal side effects.

Lipid nanoparticles (LNPs) are currently having an increasing impact on nanomedicines as delivery agents, among others, of RNA molecules (e.g., short interfering RNA for the treatment of hereditary diseases or messenger RNA for the development of COVID-19 vaccines). Despite this, the delivery of plasmid DNA (pDNA) by LNPs in preclinical studies is still unsatisfactory, mainly due to the lack of systematic structural and functional studies on DNA-loaded LNPs. To tackle this issue, we developed, characterized, and tested a library of 16 multicomponent DNA-loaded LNPs which were prepared by microfluidics and differed in lipid composition, surface functionalization, and manufacturing factors. 8 out of 16 formulations exhibited proper size and zeta potential and passed to the validation step, that is, the simultaneous quantification of transfection efficiency and cell viability in human embryonic kidney cells (HEK-293). The most efficient formulation (LNP15) was then successfully validated both in vitro, in an immortalized adult keratinocyte cell line (HaCaT) and in an epidermoid cervical cancer cell line (CaSki), and in vivo as a nanocarrier to deliver a cancer vaccine against the benchmark target tyrosine-kinase receptor HER2 in C57BL/6 mice. Finally, by a combination of confocal microscopy, transmission electron microscopy and synchrotron small-angle X-ray scattering, we were able to show that the superior efficiency of LNP15 can be linked to its disordered nanostructure consisting of small-size unoriented layers of pDNA sandwiched between closely apposed lipid membranes that undergo massive destabilization upon interaction with cellular lipids. Our results provide new insights into the structure-activity relationship of pDNA-loaded LNPs and pave the way to the clinical translation of this gene delivery technology.

Comparative analysis of lipid-peptide nanoparticles prepared via microfluidics, reverse phase evaporation, and ouzo techniques for efficient plasmid DNA delivery

AimThe current study aimed to develop enzyme-activated charge-reversal lipid nanoparticles (LNPs) as novel gene delivery systems. MethodsPalmitic acid was covalently bound to protamine being utilised as transfection promoter to anchor it on the surfaces of LNPs. Green fluorescent protein (GFP) encoding plasmid DNA (pDNA) was ion paired with various cationic counter ions to achieve high encapsulation in LNPs. Protamine-decorated LNPs were prepared by solvent injection method followed by coating with sodium tripolyphosphate (TPP) to generate a bio-inert anionic outer surface. Resulting LNPs were characterised regarding size, polydispersity, zeta potential and encapsulation efficiency. Enzyme-triggered charge-reversal of LNPs was investigated using isolated alkaline phosphatase (ALP) monitoring changes in zeta potential as well as monophosphate release. Furthermore, monophosphate release, cell viability and transfection efficiency were evaluated on a human alveolar epithelial (A549) cell line. ResultsProtamine-decorated and TPP-coated (Prot-pDNA/DcChol-TPP) LNPs displayed a mean size of 298.8 ± 17.4 nm and a zeta potential of −13.70 ± 0.61 mV. High pDNA encapsulation was achieved with hydrophobic ion pairs of pDNA with 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DcChol). Zeta potential of Prot-pDNA/DcChol-TPP LNPs reversed to positive values with a total Δ26.8 mV shift upon incubation with ALP. Conformably, a notable amount of monophosphate was released upon incubation of Prot-pDNA/DcChol-TPP LNPs with isolated as well as cell-associated ALP. A549 cells well tolerated LNPs displaying more than 95 % viability. Compared with naked pDNA, unmodified LNPs and control LNPs, Prot-pDNA/DcChol-TPP LNPs showed a significantly increased transfection efficiency. ConclusionProt-pDNA/DcChol-TPP LNPs can be regarded as promising gene delivery systems.

This study aims to investigate the effects of different non-ionic surfactants on physicochemical properties of ART niosomes. ART is a natural compound that is used as an antimalarial and chemotherapy agent in medicine. ART has low bioavailability, stability and solubility. In order to solve these problems and enhancing the efficiency of the drug, nanotechnology was used. In the present study, several niosomal formulations of ART prepared using different molar ratios of Span 60 : Tween 60 : PEG-600: ART in PBS. These three formulations were FI (1:1:0.5:0.5), FII (2:1:0.5:0.5) and FIII (1:2:0.5:0.5), respectively. The encapsulation efficiency was measured by HPLC and the drug release was evaluated by dialysis method. The cytotoxicity test was determined by MTT assay. The size, zeta potential and polydispersity index of the vesicles was measured by Zeta Sizer. Stability study was performed within two months. The MTT assay results showed that cytotoxicity effect of these formulations on MCF-7 cell line is better than C6 cell line and the FIII had the best results for both of them. The entrapment efficiencies of the formulations I, II and III were obtained 82.2±1.88%, 75.5±0.92% and 95.5±1.23%, respectively. The results of size, zeta potential and polydispersity index indicated that the size of the vesicles is below 200 nm, their surface charge is about-35 mV and they were monodisperse. Stability and release study indicated that the formulation III has the best stability and release pattern. Therefore, the use of PEGylated niosomal ART can effectively improve its therapeutic index, stability and solubility